1. Field of the Invention
This invention relates generally to methods and compositions for direct detection of specific nucleic acid sequences associated with flanking regions of chromosomal aberration breakpoints, by forming hybrids between the sequences and genetic probes, and detecting the probes. In particular aspects, the invention concerns detection of nucleic acid sequences in situ in chromosomes, and more specifically in cells, including interphase cells. Compositions of probes useful for detecting chromosomal translocations, in particular those associated with human leukemias, are also disclosed.
2. Description of the Related Art
Substantial proportions of human diseases and malformations trace their etiology, at least in part, to genetic factors. Some of these factors are present in the zygote, others occur later as somatic cells form. Detection of genetic factors associated with particular diseases or malformations provides a means for diagnosis and treatment. For some conditions, early detection may allow prevention or amelioration of the devastating courses of diseases.
One class of genetic factors are chromosomal aberrations, that is, deviations in the expected numbers and structure of chromosomes for a particular species, and for particular cell types within a species. These may be constitutive i.e. present in the zygote, or induced post-zygotically in somatic (non-germinal cells) leading to mosaicism, that is a condition where both normal and abnormal cells are present. Chromosomes are the microscopically visible entities that are composed of the genetic material and, in higher organisms such as man, proteins and RNA. The study of chromosomes is called "cytogenetics".
There are several classes of structural aberrations that may involve autosomes or sex chromosomes or both. These aberrations are detected by noting changes in chromosome morphology (band patterns). The band patterns may be only changed in one chromosome (intrachromosomal) or in more than one chromosome (interchromosomal). Normal phenotypes may be associated with these rearrangements if the amount of genetic material has not been altered, but physical or mental anomalies are expected if there is gain or loss of genetic material. Simple deletions (deficiencies) refer to loss of part of a chromosome. Duplication refers to addition of material to chromosomes. Duplication and deficiency of genetic material can be produced by simple breakage of chromosomes, by errors during DNA synthesis, or as a consequence of segregation of other rearrangements into gametes.
Translocations are interchromosomal rearrangements effected by breakage and transfer of part of chromosomes to different locations. In reciprocal translocations, pieces of chromosomes are exchanged between two or more chromosomes. Generally, the exchanges of interest are between nonhomologues. If all the original genetic material appears to be preserved, this condition is referred to as balanced. Unbalanced forms have duplications or deficiencies of genetic material associated with the exchange; that is, something has been gained or lost "in the shuffle."
One of the most exciting associations between chromosomal aberrations and human disease, is that between chromosomal aberrations and cancer. These aberrations are generally not constitutive, i.e., present in the zygote, therefore are not present in all cells--only the abnormal ones. A mosaic condition is said to exist. For example, the Philadelphia (Ph.sup.1) chromosome is an important cytogenetic finding in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL). This chromosome was originally identified as a chromosome sized slightly smaller than a "G-group" chromosome. It was believed to be a deleted chromosome until detection of a reciprocal translocation between chromosomes No. 9 and 22 was reported by Rowley. A reciprocal translocation is one caused by breakage of at least two chromosomes and reunion of the broken piece in new locations.(FIG.1) This aberration was first found to be associated with CML, but is now known to have prognostic and diagnostic value for many hematopoietic malignancies, e.g. ALL.
It is not only the translocation per se that is of clinical interest, but rather the resulting fusion of the proto-oncogene abl from the long arm of chromosome 9 with the bcr gene of chromosome 22, a consistent finding in CML. This genetic change leads to formation of a bcr-abl transcript that is translated to form a 210 kD protein present in virtually all cases of CML. This fusion can be detected by Southern analysis for bcr rearrangements or by in vitro amplification (PCR) of a complementary DNA (CDNA) transcript copied from CML MRNA. In approximately 95% of cases, the fusion gene results from a reciprocal translocation involving chromosomes 9 and 22, producing a cytogenetically distinct small acrocentric chromosome called Ph.sup.1. In the remaining cases the genetic rearrangement is more complex, and the involvement of the bcr and abl regions of chromosomes 9 and 22 may not be apparent during analysis of banded metaphase chromosomes. Southern blots, PCR, and metaphase chromosome banding analysis provide complementary, but incomplete, information on CML. They do not permit a genetic analysis on a cell by cell basis in a format in which the results can be related to cell phenotype as judged by morphology or other markers. Thus, assessment of the distribution of the CML genotype among cells of different lineage and maturity has not been possible.
As an example of the prognostic value of chromosomal aberrations, in adult ALL, the Ph.sup.1 chromosome is present in up to one-third of cases, and is associated with a high relapse rate and short survival. In pediatric ALL it is much less common, but it remains one of the few chromosomal abnormalities that continues to carry a poor prognosis in spite of newer, more intensive approaches to treatment. The accurate detection of the Ph.sup.1 is thus an important part of the diagnostic evaluation of patients with ALL.
Unfortunately, the cytogenetic diagnosis of the Ph.sup.1 chromosome in ALL has been limited. Cytogenetic analysis has a high failure rate in this disease, compared to other acute leukemias or to CML. Fewer than 70% of cases have adequately banded chromosomes at metaphases in most reports. "Banding" is a morphological pattern revealed by treating chromosomes to reveal horizontal stripes which vary in width and staining intensity and are characteristic of specific chromosomal regions. As an alternative to cytogenetic analysis, recently, newer methods of chromosomal in situ hybridization with non-isotopically labelled genetic probes have improved and extended the capabilities of cytogenetics. One of these methods is fluorescence in situ hybridization (FISH). In this method, probes are labelled with fluorescent signals that are detectable, generally by microscopic viewing of colors. Probes are nucleic acid sequences which bind to matching (homologous) sequences, e.g. on chromosomes. Although based on cytogenetic diagnosis, FISH may be performed on interphase cells as well as on metaphases, and may be applied directly to cells from either the peripheral blood or bone marrow without the need for banded karyotypes. The diagnostic utility of FISH with repetitive, centromeric probes in cases of leukemia has been demonstrated in previous studies.
FISH on interphase cells has proven to be a useful method for diagnosis and clinical management in hematologic diseases. However, much of this experience has concentrated on detecting numerical chromosomal abnormalities (single chromosome loss or gain), making use of chromosome-specific alpha satellite probes, which are highly-repetitive, unique sequences that occur within or near the centromere of chromosomes. The centromere is a constriction most readily visible at metaphase of cell division, which occurs at a characteristic location on each chromosome. The development of competitive hybridization methods to eliminate the signal from Alu-type repeats, and improvements in optics and reagents, have also made it possible to visualize single-copy genomic clones by FISH. However, the use of genomic clones is more difficult than the use of alpha satellite probes, because of lower signal intensity and high background. These difficulties would be offset if use of genomic clones produced improvements in disease assay specificity and were more flexible. Genomic clones are those that contain repeated sequences and non-coding sequences, that is DNA as it exists in the chromosome.
Some of the background for the present invention is as follows: single stranded synthetic DNA was developed with multiple sites are incorporated where fragments may be used as probes. (Stephensen, U.S. Pat. No. 4,681,840). Oncogenes are genes whose products have the ability to transform eukaryotic cells so that they grow in a manner analogous to tumor cells. Probes and methods for detecting chromosomal translocations are disclosed in EPO 181 635 (Groffen et al.)
Pinkel et al. (1986, 1988) and Gray et al. (1990) relate fluorescent-labeled probes for the cytogenetic analysis of chromosomes, and in situ hybridization of chromosomes at metaphase and interphase with whole chromosome-specific DNA.
In situ hybridization using a mixture of radioactive labelled probes c-abl and bcr sequences were employed on a CML patient sample. Although a translocation was said to be detected, Poisson analysis, a statistical procedure, was required to differentiate random from non-random silver grain distribution after autoradiography. (Bartram et al., 1987).
Benn et al. (1987) relates the molecular genetic analysis of the bcr rearrangement in the diagnosis of CML. Analysis involved Southern blots and radioactively labelled probes.
A single bcr-derived probe from which highly repetitive sequences were removed, was employed to detect the Ph.sup.1 translocation in CML. Restriction fragment length polymorphisms (RFLP) were used to identified patients affected with CML. Probes were used to map the chromosome 22 breakpoints within the bcr region by Grossman et al. (1989). Two separate bcr-specific probes were used to detect rearrangements within the bcr region. Southern blots and RFLP were employed. (Hutchins et al., 1989).
Flow cytometry has been applied to detection and characterization of disease--linked chromosome aberrations (Gray et al., 1990). There is a great need to improve methods of detecting specific chromosome aberrations. Flow cytometric requires in vitro cell culture, expensive equipment, and expertise in interpretation of statistical analyses of results. Therefore, it is not generally clinically useful.
Detection of aberrations by use of repeat sequence probes found near centromeres, generally alpha satellite probes, or whole chromosome probes not probes specific for genetic regions associated with diseases. Greater sensitivity and increased resolution is needed. Use of whole chromosome probes is generally limited to detection of aberrations that occur homogeneously in a cell population (Gray et al., 1990) and does not have the resolution to distinguish similar, but distinct breakpoints. The present invention relates methods and compositions for detection of chromosomal aberrations that need not be present in all cells of a sample. Compositions include novel probes that were specifically designed to detect the BCR-ABL fusion gene in acute and chronic leukemias e.g. CML and ALL, and to determine molecular subtypes.
Methods using a plurality of probes to provide increased sensitivity and specificity in detecting chromosomal aberrations, are also aspects of the present invention. These methods are particularly valuable in being applicable to interphase cells, thus avoiding the costly, laborious, time-consuming and often inconclusive cytogenetic analysis of metaphase chromosomes, and the expertise needed for flow cytometry. Not only are the methods of the present invention easier to use, but these methods do not require invasive or risky techniques inflicted on patients, such as bone marrow sampling. However, the methods and compositions of the present invention may also be used on metaphase chromosomes or Souther blots.